In optics, a frequently occurring problem is to collinearly combine light beams, in particular light beams of different wavelengths.
For example, in scanning microscopy, samples are often prepared with a plurality of markers, for example, a plurality of different fluorescent dyes, to simultaneously excite them with an illuminating light beam containing light of several excitation wavelengths. To produce the illuminating light beam, usually, the light beams of several lasers are combined using usually a plurality of dichroic beam splitters arranged in series. A scanning microscope having a dichroic beam combiner for infrared and ultraviolet light is known, for example, from German Published Patent Application DE 198 29 953 A1.
German Published Patent Application DE 198 35 068 A1 discloses a microscope, in particular a laser scanning microscope, with illumination over one wavelength and/or a plurality of wavelengths, where the intensity of at least one wavelength is controlled by at least one rotatable interference filter placed in the illuminating beam path, and where the at least one wavelength is at least partially reflected out of the illuminating beam path, and a plurality of filters for different wavelengths can be arranged in series in the illuminating beam path.
In scanning microscopy, the light beams of a light source are coupled into the scanning microscope and aligned to the optical path of the scanning microscope, and a sample is illuminated by the light beam to observe the reflected or fluorescent light emitted by the sample. The focus of an illuminating light beam is moved in a sample plane using a controllable beam deflection device, generally by tilting two mirrors; the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by galvanometer positioning elements. The power of the light coming from the sample is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to determine the current mirror position.
In confocal scanning microscopy specifically, a sample is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally includes a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detection or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the sample travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that point information is obtained which leads to a three-dimensional image by sequential scanning of the sample. A three-dimensional image is usually achieved by acquiring image data in layers; the track of the scanning light beam on or in the sample ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and stewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at a constant Y position, etc.). To allow the acquisition of image data in layers, the sample stage or the objective is shifted after a layer has been scanned, and the next layer to be scanned is thus brought into the focal plane of the objective.
The input coupling of the light beams for illuminating a sample into a microscope is very critical with respect to alignment, especially because the position and propagation direction of usually a plurality of light beams of different wavelengths must exactly follow the nominal optical path of the microscope. Alignment of a direct input coupling is first of all difficult, and secondly is usually not very reliable because, due to relatively long light paths, even the smallest variations in the setup lead to imperfections requiring painstaking realignment. Frequently, optical fibers are used to transport the light beams from the light source or light sources to the microscope in order to reduce the problem to an alignment of the output coupling of the optical fiber, which is in fact also painstaking, but, due to the shorter light paths, is less sensitive to misalignments. This does not solve, but at best reduce the alignment problem, and involves other difficulties, such as the variation in the polarization direction of the light beams.
The known systems for combining light beams of different wavelengths have the disadvantage of being inflexible with respect to a change in wavelength. Moreover, it is not possible to ascertain whether the combined beams actually propagate exactly collinearly. This effort is generally left to the user or to the service technician. If the light beams combined into an illuminating light beam are not substantially collinear, then abberations, in particular artifacts and variations in brightness, will occur in scanning microscopy.